1. Field of the Invention
The present invention relates to an immobilized enzyme film, a protein immobilized film and a process for forming the same, particularly to an immobilized enzyme film for biosensors and a process for forming the same. More specifically, the present invention relates to a process for forming an immobilized enzyme film or a protein immobilized film in an integrated semiconductor biosensor having such film formed on the surface of an ion-sensitive field effect transistor.
2. Description of the Prior Art
Attempts have so far been made to utilize enzymes in the field of analysis. Particularly, biosensors employing an immobilized enzyme film and various kinds of electrochemical devices are speedy, convenient and highly sensitive analytical means. Recently, a biosensor comprising a micro-electrochemical device fabricated using the silicon IC production technology and an enzyme film formed on the surface thereof has been proposed (Japanese Utility Model Publication No. 61-50262 (Japanese Utility Model Application No. 59-134995)).
As one example of such enzyme film formed on the sensitive section of such microsensor, a film formed using a mixture of an enzyme, a protein and glutaraldehyde has been used. According to this method, an immobilized enzyme film is obtained using a mixture prepared by adding glutaraldehyde to a protein solution containing an enzyme.
There is so far known a semiconductor biosensor for determining concentration of a specific organic substance contained in a solution comprising an ion-sensitive field effect transistor (hereinafter abbreviated as ISFET) having an enzyme immobilized on the surface thereof. This ISFET biosensor determines at the ISFET the concentration of a specific organic substance by detecting a change in the hydrogen ion concentration in the solution which occurs when the specific organic substance undergoes a chemical reaction by the catalytic action of the enzyme. As such immobilized enzyme film having selectivity, urease immobilized films for urea detection and glucose oxidase immobilized film for glucose detection are known.
A process for producing such biosensor has been proposed by Kuriyama, Nakamoto et al., in which glutaraldehyde is used as a crosslinking agent for an albumin immobilized film or an immobilized enzyme film to pattern a glutaraldehyde-albumin crosslinked film and a glutaraldehyde-albumin-enzyme film on a wafer by photolithography (Japanese Patent Application Nos. 59-209165 and 60-194333). Meanwhile, Miyamoto proposed a method in which such film is formed by spin coating with cooling so as to obtain a film having a uniform thickness (Japanese Patent Application No. 63-325216).
As described above, when an immobilized enzyme film is formed according to the prior art technique, the resulting film assumes hydrophobicity since the film is crosslinked by glutaraldehyde, so that the water content after drying will be extremely low to increase internal strain of the film and give stress to the enzyme retained in the film, in turn, accelerating deactivation of the enzyme, disadvantageously.
On the other hand, in the case of a glutaraldehyde-crosslinked enzyme film having no protective film such as of saccharide, a conformational shift occurs in the enzyme due to the shrinkage of the film during film formation or determination, inevitably causing deactivation of the enzyme. Besides, the crosslinking reaction to be effected by the glutaraldehyde must be controlled by cooling so as to obtain a film having a uniform thickness.